Digital flow cytometer and method

ABSTRACT

A system and method for testing for microbial contamination using a digital flow cytometer comprising an illumination source, a fluorescence detector, a digitizing device, and a digitized signal transferring device.

This application is a 371 of PCT/U.S. 98/25723 filed Dec. 11, 1998, andalso claims benefit of Provisional No. 60/069,528 filed Dec. 12, 1997.

TECHNICAL FIELD

The present invention is addressed to an improved system for testing formicrobial contamination in industrial products.

BACKGROUND ART

Adequate supervision of the safety of industrial products dependsheavily on the detection of microbial contamination of the product. Thismicrobial contamination can exist in a wide variety of industrialproducts including food, drinking water and health and beauty aids. Anormal approach to detecting microbial contamination involves testswhich depend upon the incubation of a sample taken from the product in amedia which is suitable for the growth of micro-organisms. This approachinvolves the growth of microbes to ensure their viability and at thesame time the multiplication of signals in order to simplify theirdetection. However, in many instances it requires several days toperform the test which imposes severe delays in manufacturing andinventory cost This is extremely critical in cases where the product islabile and the result of microbial testing is a shorter shelf life.Evidently then it is extremely important to develop ways of performingthe microbial testing in a rapid manner.

Rapid testing methods which detect microbes without requiring amultiplication by growth usually involve labels which have beendeveloped to effectively mark any viable micro-organism through the useof luminescence or fluorescence.

The general drawback with these methods has been the limit placed on theentire operation by the effectiveness of the instrumentation which mustbe capable of picking out the labeled microbe from other interferingsignals with sufficient reliability to be useful in everyday practice.Practical use of such systems requires the ability to have a sensitivityto contamination which is very high detecting 100 or less microbes permilliliter of product. There is also a simultaneous requirement to havean extremely low false alarm rate of less than 1% for example.

One currently used instrumentation which has been attempted as asolution is a fluorescent flow cytometer wherein a diluted sample passesthrough a laser beam and photodetectors are used to note anyfluorescence. Such a device when coupled with a prior device forfluorescently labeling each individual viable microbe, appears to be auseful tool in this area of microbial contamination detection. However,in spite of many attempts, this technology has not proved practical fora wide class of industrial products primarily due to the limitations inthe sensitivity and/or specificity which arises.

The above discussed instrumentation and fluorescent labeling generallyfalls into two categories or two approaches to labeling thecontaminating microbes. They both depend upon the action of a ubiquitousenzyme within the microbe-organism to create an optical signal. In oneinstance the resultant is a luminescent reaction while in the otherinstance the microbe is rendered fluorescent. The applicable use foreither of these labeling method is limited by either the sensitivity ofthe luminescence method, so that enough light is not generated by asingle microbe to be detected, or the specificity with regard to thefluorescent methods wherein any light from the labeled microbe cannot bedistinguished from background fluorescent sources.

The automation of fluorescent methods of rapid microbiology yields twocurrently used approaches. In the fluorescent flow cytometry approach, adiluted suspension of a product to be tested is interrogated by passingit through a laser spot and detecting the resultant fluorescence oflabeled microbes. On the other hand in the method known as the solidphase cytometry for instance as described in U.S. Pat. No. 5,663,057, asample of a liquid product is passed through a membrane filter withsufficiently small pore size to retain any microbes and the filter issubsequently scanned by laser beam to detect any labeled microbes.

These two methods use different sampling means and address differentproducts. For example some samples may not be filterable and thus cannotbe used with the solid phase cytometer. Furthermore the level ofperformance which measures the sensitivity to the contaminating elementwhich is obtained from the fluorescent flow cytometry is different fromthe solid phase cytometry. In fact, the solid phase cytometry isconsistently more effective at detecting contamination. This differenceis not due to the relative sensitivity but instead is due to therelative specificity. That is, both detectors have sufficientsensitivity to respond to a single microbe but the solid phase cytometeruses a set of sophisticated discriminators which are applied to adigitized waveform representing the fluorescent signal and thesediscriminators are based on the relative amplitude and detailed phaseshape of individual fluorescent signals obtained at two or more opticalwavelengths. It is for this reason that the solid phase cytometer ismore effective at detecting contamination than the currently usedfluorescent flow cytometers.

This difference in discrimination ability occurs because, whencontrasted with the solid phase cytometers, the commercial fluorescentflow cytometer employ analog circuits which produce the feature valuesof the pulse waveform resulting from the particle fluorescence. This useof analog pulse processing limits the features which can be measured totheir pulse integral, pulse height and pulse width. Thus, a significantamount of relevant information concerning the shape of the waveform islost.

However it must be pointed out that, although digital processing hasbeen applied to flow cytometers, its use has generally been limited bydata processing speed. That is, when the sampling rate is madesufficiently high to obtain the required resolution for analyzing asingle pulse, the processing system cannot keep up with the random pulsearrival rate. That is, the lowest sampling rate still produces an outputfor which continuous sampling is not possible. In order to resolve thisissue a compromise in digital resolution is usually used and thus acompromise in the potential performance.

Yet another approach to obtaining additional information regarding thevariation in fluorescent signals over time has been to use an array ofdetectors (linear CCD) which extend along the particle trajectories. Inthis method, the signal from each detector is processed in an analogmanner and the results are combined to obtain a signal waveform. Whilethis signal has been shown useful to measure the fluorescent decay it isa complicated system which must be precisely set up and it is limited bythe relative sensitivity of adjacent detectors.

DISCLOSURE OF THE INVENTION

Accordingly one object of this invention is to provide a novel samplingstrategy with a flow cytometer instrument in order to mitigate problemswith data processing by interrupting the sampling interval in a digitalflow cytometer periodically to allow data processing to keep up with thedigital sampling rate.

It is a further object of the present invention to decouple the peaksampling rate from the average data transfer capacity and to maintainsteady state conditions for the entire measurement, in order to providea sample which will be representative even with the interrupted sampleinterval and wherein the sample size is determined by the cumulativesample interval.

It is a further object of the present invention to provide a techniquewhich is particularly useful for a rapid microbiology wherein it iscrucial to separate fluorescent signals originated from labeled microbesfrom those produced either by induced auto-fluorescence, freefluorescent dye or non-specific labeling.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 illustrates a structural diagram of a conventional opticalarrangement for use in the present invention;

FIG. 2 is a functional block diagram of the digital flow cytometer ofthe present invention;

FIG. 3 is an illustration of the sampling scheme used in the digitalflow cytometer of the present invention;

FIG. 4 illustrates a fluorescent pulse intensity profile over time;

FIG. 5 is a flowchart illustrating the operation of the digitaldiscrimination according to the present invention;

FIG. 6 is a flowchart illustrating the feature detection methodaccording to the present invention;

FIG. 7 is a flowchart of the color ratio discrimination method accordingto the present invention;

FIG. 8 is a flowchart illustrating the Gaussian discriminator methodemployed in the present invention;

FIG. 9 is a flowchart illustrating operation of an analog discriminator;and

FIG. 10 is a schematic illustration of a general purpose computer 300programmed according to the teachings of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Referring now to the drawings, wherein like reference numerals designateidentical or corresponding parts throughout the several views and moreparticularly to FIGS. 1 and 2 thereof, there is illustrated a layout ofthe optical structure 30 utilized for the digital flow cytometer and itsoperation in the present invention. The digital flow cytometer of thepreferred embodiment is illustrated in the optical structure of FIG. 1wherein light from an argon ion laser 10 is used to illuminate a flowstream of a vertical rectangular quartz flow cell 6. This flow streamcontains diluted specimen 34 which have been previously labeled with afluorescent dye sensitive to microbes and excited at 488 nmcorresponding to the wavelength of the argon ion laser. The excitationcan occur, for example, by using a substrate to the ubiquitousnon-specific esterases which converts carboxyfluorescein to afluorescent state. The position of the output beam from the laser 10 iscontrolled by the lateral position adjustment 11 and the verticalposition adjustment 12. The positioning of the flow cell is controlledby the x position adjustment 13 and the y position adjustment 14. Theoutput of the laser is focused by the optics 15 which is a crosscylinder lens providing an elliptical spot when focused at the center ofthe flow cell 6. The flow stream passes the illuminated spot at avelocity of approximately 8 meters per second with the illuminated spothaving a bi-variate Gaussian intensitive profile with a smallerdimension of between 10 and 30 microns in the direction of flow and alonger dimension of between 60 and 100 microns orthogonal to the flowdirection.

The fluorescence which results from the laser on the specimen isdetected by an optical arrangement which includes an objective lens 17positioned at a 90° angle to the illuminated beam. The detectedfluorescent is split by a combination of mirrors onto two separate PMT(photo multiplier tube) detectors 24 and 25. The path between theobjective lens 17 and the photo multiplier tubes 24 and 25 in anillustrative embodiment of FIG. 1 utilizes dichroic mirror 18, relaylens 19, non-selective mirrors 20 and 20′ and a second imaging lens 21which passes the image to a dichroic mirror 22 (having a long pass above615 nm) and finally to another non-selective mirror 20″. The redsensitive photo multiplier tube 24 receives the image from the mirror20″ and feeds it to a transimpedance preamplifier 26′ while the photomultiplier tube 25 receives the image from the dichroic mirror 22through the bandpass filter (510-540 nm). The outputs of each of thephoto multiplier tubes 24 and 25 are fed to transimpedance preamplifiers26, 26′ in order to convert from current to voltage.

The signal train from the two photo multiplier tubes 24 and 25 and thepreamplifiers 26 and 26′ are then converted through respective a/dconverters 40 and 40′ as shown in FIG. 2. These a/d converters are12-bit converters which function at a sampling rate of 5 MHZ. The outputof the A/D converters are fed to a transfer device 52 which functions toprovide interruption by way of the time gating device 56 according to aparticular interruption scheme.

The characteristics of a typical fluorescent signal amplitude curve, asshown in FIG. 4, are obtained from a fluorescent labeled microbe underthe conditions described with respect to FIG. 1.

Transfer and processing of the digital signals according to the presentinvention is illustrated in FIG. 5. Complete sampling data 251 from eachof the two photo multiplier tubes 24 and 25, during a total samplingtime of approximately 18 milliseconds (T of FIG. 3), is gated by timegate 56 in FIG. 2, into a temporary storage buffer 252. Subsequently,when a shorter sampling interval (t of FIG. 3) is complete, thethreshold features are determined at 254 as will be described withrespect to FIG. 6. Then the feature samples are transferred forcomputation at 255 and the total sample is tested for completion to seewhether the total sampling time T (FIG. 3) has been complete asindicated at 256. If the total sampling time has not been completed,then another interval of two milliseconds is tested until the totalsample time has been completed at which time the feature passdiscriminators 257 determine whether the feature is to be added to thecount or rejected. Furthermore, after each sampling interval, the dataflow is interrupted (g of FIG. 3). During the shorter sampling interval(t) a moving threshold algorithm, as detailed in the flowchart of FIG.6, is used to isolate regions (features) in which the fluorescent signalexceeds expectations and the sample points from each feature are thentransferred into a computer for further discrimination. As shown in FIG.6 a threshold is determined at 310. If a signal is above a currentlyfrozen threshold as indicated at step 320, then the sample is added tothe current feature as indicated at 330. On the other hand, if thesignal is not above the currently frozen threshold, the feature isclosed as indicated at 340 and added to the background level calculation360. If the current threshold is not frozen at 350, then if the signalis above the threshold then the threshold is indeed frozen at 370 with asubsequent storing of a new feature at 380. If the signal is not abovethe unfrozen threshold as determined at 350, then the signal is added tothe background level calculation 360. When this process is complete anew measurement interval (t) is begun. This alternation of samplinginterval (t) and interrupted time (g) continues until the end of thetotal sampling time (T). This interval is typically 3,000 cycles. Thestored features are then analyzed by a set of discriminating algorithms60.

This system resulted from a comparison of the differences between solidphase cytometry and flow cytometry which initially indicated that thepeak data transfer rate was essentially the same between the twosystems. This unexpected result led to a realization that the differencebetween the two systems was in the average data transfer rate. In thesolid phase system, the scanning is performed through the use of drivenmirrors so that the initiation of each scan line was controlled by asignal. Typically, the scan retrace time was used to buffer the datatransmission rate. A new scan in the solid phase system was only startedwhen the processor had caught up with data from the previous scan.Because the entire membrane was scanned no data was lost but thescanning period was variable.

In contrast, in the flow cytometer system, the diluted specimen wasanalyzed for a predetermined interval of time. This interval isinitiated after steady flow condition is established with the dilutedspecimen passing the detection station. It is assumed that, provided thesteady state conditions are maintained during the sampling interval, arepresentative sample has been obtained. The size of the analyzed sampleis proportional to the length of the sampling interval.

The present invention is based upon the employment of the uniquesampling strategy discussed with respect to the FIG. 3 for use with aflow cytometer instrument in order to mitigate the problem with respectto data transfer. More specifically, the interruption (g) in thesampling interval in a digital flow cytometer occurs periodically inorder to allow the data transfer and processing to keep up with thedigital sampling rate. The effect is to decouple the peak sampling ratefrom the average data transfer capacity. As long as the steady stateconditions are maintained for the entire measurement period, the samplewill still be representative and the sample size will be determined bythe cumulative sample interval. This system can be used with any degreeof digital resolution. In order to use higher resolutions, either therate or duration of the interruptions need to be increased so that thesame average data transfer rate can be maintained. Thus, with higherresolution, the cumulative sample interval must be extended. Thistechnique is especially useful when the digital flow cytometer is usedfor Rapid Microbiology. In this case, it is crucial to separatefluorescent signals originating from labeled microbes from thoseproduced either by induced auto-fluorescence or from free fluorescentlabel.

One of the keys to this discrimination is the size of the particle whichis represented in the shape of the fluorescent pulse (intensity vstime). Microbes are very small compared to the laser spot. Thus, whenthey pass the illuminated region, the shape of the resultant fluorescentpulse accurately reflects the Gaussian distribution of intensity asshown in the example of FIG. 4. This is important because the Gaussiandistribution of intensity is a characteristic of the laser beam. On theother hand, larger objects generally display extended non-Gaussianintensity profiles. Free fluorescent label causes variations influorescent intensity which are essentially random and thus are alsonon-Gaussian. Therefore, a discriminator which analyzes the fluorescentintensity waveforms and compares them to a Gaussian distribution is aneffective means of discrimination of the fluorescence from the microbialparticles.

Effective discrimination is also affected by the fluorescent lifetime.Fluorescent labels can be chosen with fluorescent lifetimes which aresubstantially longer than characteristic auto-fluorescence. In thiscase, by comparing the fluorescent pulse to the illuminating intensity(as measured by the scattering of illuminating light) in time, thedifferences may be related to the fluorescent lifetime. Shortfluorescent lifetimes of less than 10 nanoseconds may be distinguishedfrom the longer lifetimes. Finally, effective discrimination is relatedto the time correlation between fluorescent intensity at twowavelengths. In the case of a discrete particle such as a labeledmicrobe, the peak intensity in two channels is highly correlated while,on the other hand, the random fluctuations resulting from freefluorescent labeling in the specimen is not well correlated between thetwo fluorescent channels.

Therefore, the stored features in the discriminating feature section 60of FIG. 2 are analyzed by a set of discriminating algorithms including acolor ratio discrimination detailed in FIG. 7 and a Gaussian profilediscrimination detailed in FIG. 8. With respect to the color ratiodiscrimination of FIG. 7, the ratio of each sample point in the greensignal is compared to the signal level in the red channel and a ratio isformed. This ratio is compared to the ratio expected from the spectrumof the fluorescent dye used in labeling bacteria. If the ratio exceedsthe expected limit, the feature is rejected as being consistent withauto-fluorescence as opposed to specific labeling. More particularly,the red/green signal ratio at 110 is compared with an acceptable ratioat 120 if there is also an event feature present in green channel 130.If the ratio is within acceptable boundaries and if the feature event ispresent in the green channel, then the event is retained at 140. On theother hand, if there is no event feature present in the green channel orthe ratio between the red and green channel signal is outside ofacceptable ranges, the event is rejected at 150.

With respect to the other discrimination algorithm shown in FIG. 8, theGaussian profile discriminator fits sample points in each feature to aGaussian curve. Because the laser beam intensity is a Gaussian profile,fluorescent signal from objects which are very small compared to thedimensions of the laser spot will reproduce a Gaussian intensity profilewhile larger objects will be typically non-Gaussian in their intensityprofile. Because all labeled microbes can be expected to be very smallcompared to the laser spot, those features having non-Gaussian profiles(indicated by the measure of “goodness”) are rejected. Moreparticularly, as shown in FIG. 8 after a feature is selected at 210, itis fit into a Gaussian in order to sample points at 220 and then thetest fit is determined at 230 while its “goodness” criteria issubsequently checked as to whether it is a non-Gaussian profile at 240.If it is a non-Gaussian profile, it is rejected at 250 while on theother hand, if it is a Gaussian profile fit, then the feature isretained as being a labeled microbe at 260.

In addition to the above discussed key discriminators of FIGS. 7 and 8,a number of other discriminating criteria may be applied to the storeddata in order to enhance discrimination between fluorescent labeledmicroorganisms and other spurious interfering signals. These may beapplied either one at a time or in combination. For example, the featurelength which indicates a number of digital samples in the pulse at thehalf power point may be used as a discriminator as well as the specificintensity which is a measure of the peak intensity divided by thefeature length. Furthermore, the symmetry, which is the rate of rise offluorescence compared to fluorescent decay, and the correlationcorresponding to the peak intensity in one or more fluorescent channelsmay also be utilized as a way of further enhancing the discrimination.After features associated with each specimen have passed through thediscriminating criteria they are then enumerated and the resultsdisplayed as a count at 70 shown in FIG. 2.

In order to analyze the improvement brought about by the digital flowcytometer of the present invention it is helpful to begin by recognizingsome of the key times involved in the process of analyzing approximately100 microliter of a specimen as detailed in Table 1.

TABLE 1 EVENT OR PROCESS TYPICAL TIME PARTICLE TO PASS LASER SPOT 1.2usec EACH DIGITAL SAMPLE 0.2 usec SAMPLING INTERVAL (typical) 18,000usec INTERRUPT INTERVAL (approx) 10,000 usec TOTAL MEASUREMENT PERIOD 43seconds

Based on the events and the typical occurrences of Table 1, anexperiment was conducted to demonstrate the digital processing scheme ofthe present invention in comparison with an analog processing of thesame samples. For the purpose of this experiment, analog signals weresplit at the output of each photo multiplier tube with one set analyzedby the digital scheme described above and the other analyzed by atypical analog processing algorithm. A typical analog processingalgorithm and its logic is shown in FIG. 9 wherein a green channelsignal at 411 is tested at 412 to determine whether the signal intensityfalls between predetermined values. If it does not then the signal isrejected at 413. If the green channel signal does fall between thepredetermined values, the red channel is triggered at 414 and thechannel ratio is calculated at 415. If this calculated ratio intensityfalls between other predetermined values as indicated at 416, then oneis added to the total count at 418. If does not fall within thesepredetermined values, then the signals are rejected at 417.

A comparison of the discrimination results from the digital flowcytometer time scheme and subsequent discrimination determination of theFIGS. 7 and 8, of the present invention when compared with the analogfeature detection of FIG. 9 is displayed in the Table 2.

TABLE 2 DISCRIMINATOR ANALOG DIGITAL GREEN PEAK INTENSITY X X GREEN/REDPEAK INTENSITY RATIO X X GREEN/RED INTEGRAL RATIO — X GAUSSIAN FIT — XFEATURE LENGTH — X

This Table 2 reveals the discrimination capabilities of the digitalsystem when compared with the analog system. The results were comparedfor a number of test specimens as well as some specimens typicallyencountered in testing industrial products for microbial contamination.Results were also compared for detection of specifically labeledfluorescent beads of approximately 0.2 micron diameter which were usedto stimulate bacteria. The results obtained are tabulated in Table 3where it can be seen that although the results are equivalent when thespecimen consist of simply clean water or water spiked with fluorescentparticles, the digital processing scheme is much more effective ineliminating spurious counts from auto-fluorescence present in unlabeledspecimens without compromising detecting of bacteria.

TABLE 3 ANALOG DIGITAL EXPECTED COUNT COUNT SPECIMEN COUNT (AVERAGE)(AVERAGE) WATER BLANK 0 2 2 (2 specimens) FLUORESCENT 400 433 414 BEADSIN WATER (3 specimens) E-COLI IN WATER >100 9230 109 E-COLI INWATER >100 364 318 E-COLI IN WATER >100 3218 261 CHOCOLATE DRINK 0 958 3UNLABELED (9 specimens) FLUORESCENT 300 388 278 BEADS IN CHOCOLATE DRINK(6 specimens) COSMETIC 0 1154 0 UNLABELED (5 specimens) FLUORESCENT 500952 519 BEADS IN COSMETIC (approx 500/specimen) E-COLI IN COSMETIC >10095 109

Another experiment determined discrimination from free fluorescentlabeling. In this experiment the analysis of free fluorescein insolution was compared to the signals obtained from fluorescein labeledbeads. The intensity of free fluorescein was adjusted so that the sameaverage fluorescent signal was obtained. Because the color of the freefluorescein and the beads was the same, approximately the same ratio ofintensity was obtained between the two fluorescent channels. Table 4illustrates the comparison between the two specimens when the additionaldigital discriminators are considered:

TABLE 4 FITC DISCRIMINANT FREE FLUORESCEIN LABELED BEADS AVERAGE PEAK314 186 (ADC COUNTS) COLOR RATIO 0.8 0.8 PULSE HALF WIDTH 24 12(SAMPLES) CORRELATION 0.289 0.90 (PMT1 vs PMT2) GAUSSIAN FIT 776 537(ERROR) SPECIFIC INTENSITY 12.7 23.9

When discrimination parameters were set on the basis of the last fourdiscriminators alone, an acceptance of greater than 60% was obtained forthe FITC labeled beads while only 0.04% of the signals from the freefluorescein were accepted. Thus, a discrimination ratio of greater than1500 between beads and free fluorescein was obtained for signals havingsimilar integrated pulse amplitude and similar color ratio. Thus, theseresults illustrate the clear improvement resulting from the additionaldigital discriminators.

The present invention includes a computer program product which is astorage medium including instructions which can be used to program acomputer to perform processes of the invention. The storage medium caninclude, but is not limited to, any type of disk including floppy disks,optical discs, CD-ROMs, and magneto-optical disks, ROMs, RAMs, EPROMs,EEPROMs, magnetic or optical cards, or any type of media suitable forstoring electronic instructions.

FIG. 10 is detailed schematic diagram of a general purpose computer 300which could be used to accomplish the functions of the transfer device52, the digital signal processor 54, the time gating 56 and thediscriminator 60 of FIG. 2. In FIG. 10, the computer 300, for example,includes a display device 302, such as a touch screen monitor with atouch-screen interface, a keyboard 304, a pointing device 306, a mousepad or digitizing pad 308, a hard disk 310, or other fixed, high densitymedia drives, connected using an appropriate device bus, such as a SCSIbus, an Enhanced IDE bus, a PCI bus, etc., a floppy drive 312, a tape orCD ROM drive 314 with tape or CD media 316, or other removable mediadevices, such as magneto-optical media, etc., and a mother board 318.The motherboard 318 includes, for example, a processor 320, a RAM 322,and a ROM 324, I/O ports 326 which are used to couple to the imageacquisition device 200 of FIG. 1, and optional specialized hardware 328for performing specialized hardware/software functions, such as soundprocessing, image processing, signal processing, neural networkprocessing, etc., a microphone 330, and a speaker or speakers 340.

Stored on any one of the above described storage media (computerreadable media), the present invention includes programming forcontrolling both the hardware of the computer 300 and for enabling thecomputer 300 to interact with a human user. Such programming mayinclude, but is not limited to, software for implementation of devicedrivers, operating systems, and user applications. Such computerreadable media further includes programming or software instructions todirect the general purpose computer 300 to perform tasks in accordancewith the present invention.

The programming of general purpose computer 300 may include a softwaremodule for digitizing, transferring, interrupting, signal processing anddiscriminating as detailed in FIG. 2. Alternatively, it should beunderstood that the present invention can also be implemented to processdigital data transferred by other means.

The invention may also be implemented by the preparation of applicationspecific integrated circuits or by interconnecting an appropriatenetwork of conventional component circuits, as will be readily apparentto those skilled in the art.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed is:
 1. An apparatus for detecting and countingfluorescent particles, comprising: an illuminating source providing afluorescence stimulating beam; means for supplying a suspensioncontaining fluorescently labeled particles to pass through said beam ata high velocity; means for detecting fluorescence of said particles andconverting said fluorescence into electrical signals; means fordigitizing said electrical signals wherein said digitizing occurs at arate sufficient to reproduce an intensity profile of an individualfluorescent particle passing said stimulating beam; means fortransferring said digitized signals and means for providinginterruptions of said transferred digitized signals and outputtingdigitized data to a digital signal processor, wherein a duration of eachof said interruptions is a function of the operating speed of saiddigital signal processor in order to facilitate the transfer of digitalinformation whereby the interruptions cause transfer and processing ofdata to be matched with the digital sampling rate of the digitizingmeans; means for analyzing digitized data output from said signalprocessor whereby particles are distinguished from background noise. 2.The apparatus according to claim 1, wherein said means for detectingfluorescence and converting said fluorescence includes at least twophotomultiplier tubes.
 3. The apparatus according to claim 1, whereinsaid means for transferring includes a storage buffer for continuousstorage at high rates for a predetermined measurement interval.
 4. Theapparatus according to claim 1, wherein said means for analyzingincludes a moving threshold discrimination means for discriminatingfluorescence features which functions to provide a discriminativefluorescence feature when a fluorescence intensity level exceeds a localaverage value.
 5. The apparatus according to claim 2, further includinga means for providing a comparison ratio of outputs of said at least twophotodetectors.
 6. A method for detecting and counting fluorescentparticles, comprising the steps of: providing a suspension containingfluorescently labeled particles at a high velocity; providing afluorescence stimulating beam; detecting resulting fluorescence of saidparticles and converting said fluorescence into electrical signals;digitizing said electric signals at a digitizing rate sufficient toreproduce an intensity profile of an individual particle in saidsuspension as it passes said stimulating beam at high velocity andoutputting digitized signals; transferring said outputted digitizedsignals and intermittently interrupting said digitized signals toprovide digital data; performing a digital signal processing on saiddigital data wherein the interrupting of said digitized signals occursfor a period of time which is a function of a speed of operation of saiddigital signal processing in order to facilitate the transfer of digitalinformation whereby the interruptions cause transfer and processing ofdata to be matched with the digital sampling rate of the digitizing ofsaid electric signals; analyzing data output from said digital signalprocessing whereby particles are distinguished from background noise. 7.Method according to claim 6 wherein said step of detecting fluorescenceand converting said fluorescence into electrical signals include the useof at least two photomultiplier tubes to provide two different wavelengths.
 8. The method according to claim 6 wherein said step oftransferring includes the step of storing at high rates for apredetermined measurement interval.
 9. The method according to claim 6wherein said step of analyzing includes a step of discriminatingfluorescent features by moving threshold which functions to provide adiscriminating fluorescent feature whenever a fluorescent intensitylevel exceeds a local average value.
 10. Method according to claim 7further including the step of providing a comparison of outputs of saidat least two photodetectors.